Application of HindIII and EcoRI Restriction Endonucleases in Identifying and Diagnosing Cystic Fibrosis caused by the CFTR ∆F508 Mutation

Application of HindIII and EcoRI Restriction Endonucleases in Identifying and Diagnosing Cystic Fibrosis caused by the CFTR ∆F508 Mutation

Ingrid Schoonover

November 1, 2019

Cellular Biology Lab

Abstract

Cystic fibrosis (CF) is caused by a mutation on the CFTR protein; preventing transport of salts across epithelial cell surfaces leading to mucus hyperproduction and eventually death. The purpose of this experiment was to determine if a 3-year old patient has cystic fibrosis. The hypothesis stated that Jeff would have cystic fibrosis caused by the CFTR ∆F508 mutation. It was predicted that Jeff’s DNA and the positive/negative controls would be cut by EcoRI two times producing three bands with the sizes 2,150 bp, 2,150 bp, and 4,700 bp and that Jeff’s DNA and the positive control would be cut by HindIII once producing two bands with the sizes 7,200 bp and 1,800 bp. The experiment used EcoRI and HindIII restriction endonucleases in RFLP analysis and visualized the results with electrophoresis on an agarose gel, the molecular size of these DNA fragments was calculated from an equation produced from a standard curve graph. The DNA of a patient with the CFTR ∆F508 mutation was included as the positive control and the DNA of a patient without the CFTR ∆F508 mutation was included as the negative control. The results of the experiment showed that all of the DNA samples cut by EcoRI produced the similar-sized DNA fragments, and Jeff’s DNA and the positive control were cut nearly identically by HindIII, while the negative control was cut differently. These results led to the acceptance of the hypothesis, meaning that Jeff was diagnosed with cystic fibrosis caused by the CFTR ∆F508 mutation.

Introduction

 

In healthy individuals, the body responds to respiratory infections by increasing mucus levels in the lungs and respiratory tract. However, this mucus can also trap bacteria and foreign material so the cystic fibrosis transmembrane conductance regulator (CFTR) protein transports salts including chlorine ions, bicarbonate ions, and anions across epithelial lung cells to hydrate the lungs and clear away excess mucus (Gentzsch, 2018; Southern et al. 2018).

However, there are over 2000 mutations of the CFTR gene that can result in the deadly disease Cystic Fibrosis (Southern et al. 2018). These CFTR mutations are grouped into five different classes based on how they affect the CFTR protein: Class I mutations produce a dysfunctional CFTR protein by adding an early stop codon to the genetic sequence, Class II mutations produce an abnormal CFTR protein most of which is degraded by the cell before it reaches the lung lining, Class III mutations produce CFTR proteins that cannot transport ions across epithelial lung cells, Class IV mutations produce impaired CFTR proteins that have a difficult time transporting ions across epithelial lung cells, Class V mutations produce a functional CFTR protein but in lower numbers than usual so this reduces its efficiency (Southern et al. 2018).

The most common of the CFTR mutations, responsible for 90% of Cystic Fibrosis cases (Cooney, 2018) is a Class II defect called the ∆F508 mutation, this specific mutation occurs when phenylalanine is deleted at position 508 of the CFTR gene (Suaud et al. 2011). This is a very common and lethal autosomal recessive disease that affects 1 in 2000 North Americans or 70,000 individuals globally (Cutting, 2015; Southern et al. 2018). This disease is lethal due to a dysfunctional CFTR protein which means that mucus builds up in the lungs and pancreatic ducts which traps bacteria, weakens the immune system, damages organs, causes inflammation, and often leads to diabetes and/or malnutrition, usually, patients die from respiratory failure (Cutting, 2015; Southern et al. 2018). Fortunately, there are several treatments to mitigate the effects of this disease and extend the life of affected individuals, and since cystic fibrosis is only caused by mutations on the CFTR gene it is relatively easy to test and diagnose patients (Cutting, 2015).

These single-gene autosomal recessive disorders (Cutting, 2015) create a type of genetic variation of the CFTR gene within a population called polymorphism (Pare, 2012). One of the most common ways for diagnosing single-mutation polymorphisms is with a technique called restriction fragment length polymorphism (RFLP) analysis, which uses restriction endonucleases to cut DNA sequences into fragments at specific sites to aid researchers in identifying genetic variations (Loenen et al. 2014; Sapienza, 2012). Restriction endonucleases are enzymes that are naturally produced in several species of prokaryotes but have many applications in laboratory genetic experiments (Pingoud et al. 2014). Restriction endonucleases or REases are grouped into four categories (Type I, Type II, Type III, and Type IV); the most commonly used in genetic testing are Type II REases which function by cleaving the phosphate bands on or near the recognition sequence in the DNA, this process produces consistent DNA fragments (Pingoud et al. 2014; Sapienza, 2012).

Two of the most understood of the Type II REases include EcoRI and HindIII: EcoRI which was discovered from Escherichia coli and HindIII was discovered from Haemophilus influenzae rd (Pingoud et al. 2014). Specifically, EcoRI and HindIII locate their specific staggered nucleotide recognition sequences and cleave the phosphate bonds between the nucleotides: EcoRI recognizes GAATTC and cleaves the phosphate bond between the G and A, additionally, it will recognize and cut any sequences that differ by one base-pair, HindIII recognizes AAGCTT and cleaves the phosphate bond between the two A’s (Loenen et al. 2014; Sapienza, 2012). Additionally, since these two REases produces symmetrical staggered cuts, the DNA fragments can anneal to their complementary strand; this has been exploited for genetic cloning applications (Loenen et al. 2014). When EcoRI is added to the blood or saliva DNA sample of a patient either with or without the CF∆F508 mutation, the EcoRI cleaves the CFTR gene twice to produce three fragments that are 2,150 bp, 2,150 bp, and 4,700 bp. However, HindIII produces different results when added to the blood or salvia DNA sample of patients with and without the CF∆F508 mutation. In patients without the CF∆F508 mutation, HindIII cleaves the CFTR gene twice to produce three fragments that are 1,500 bp, 5,700 bp, and 1,800 bp. On the other hand, in patients with the CF∆F508 mutation, HindIII does not identify the recognition sequence at 1,500 bp because of the phenylalanine deletion at position 508 messes up the rest of the sequence, so instead HindIII cleaves the CFTR gene only to produce two fragments that are 7,200 bp and 1,800 bp. The reason that the HindIII produces different results in patients with and without the mutation, while EcoRI produces identical results is that HindIII is more specific and can only identify exact recognition sequences whereas EcoRI is slightly less specific and can identify slightly varied sequences. The results of RFLP analysis can be analyzed with electrophoresis on an agarose gel, a fluorescent dye is added to the DNA samples so that it fluoresces under a UV transilluminator to reveal the distance that the fragments have traveled. The distance that the samples travel is proportional to their length, therefore, the distances that marker DNA fragments travel can be used to create an equation from a standard curve that will use the distances that the other DNA samples traveled to determine their molecular size.

This experiment is going to be testing an adopted 3-year old named Jeff for Cystic Fibrosis caused by the CFTR ∆F508 mutation. To determine if Jeff has cystic fibrosis his DNA will undergo RFLP analysis with the restriction endonucleases EcoRI and HindIII. A positive control (patient with the ∆F508 mutation) and negative control (patient without the ∆F508 mutation) will also undergo RFLP analysis with EcoRI and HindIII so that Jeff’s results can be compared to something. The results of the RFLP analysis will be visualized by electrophoresis on an agarose gel and then the equation produced from a standard curve will be used to calculate the molecular sizes of the DNA fragments after they are cut by the REases. Determining if Jeff has cystic fibrosis and what type of mutation his cystic fibrosis is caused by is important so he can receive the best treatment so that he can live a pain-free and extended life.

It is hypothesized that Jeff has cystic fibrosis because he displays several of the symptoms associated with cystic fibrosis including wheezing, crackling, persistent cough, greasy stools, and a runny nose. Furthermore, because cystic fibrosis caused by a CFTR ∆F508 mutation is an autosomal recessive disorder it is possible that both his parents were carriers of the mutation and that he inherited two mutated alleles, this explains why his parents would not have a medical history of cystic fibrosis.

If this hypothesis is correct and Jeff does have cystic fibrosis caused by the CFTR ∆F508 mutation, then it is predicted that when his DNA will be cut by EcoRI two times to produce three bands that have molecular weights of 2,150 bp, 2,150 bp, and 4,700 bp and that his DNA will be cut by HindIII once to produce two bands that have molecular weights of 7,200 bp and 1,800 bp. Also, the controls (patients with and without the ∆F508 mutation) will also be cut by EcoRI two times to produce three bands that have molecular weights of 2,150 bp, 2,150 bp, and 4,700 bp. Therefore, Jeff’s DNA and the patients with and without the ∆F508 mutation will have bands with identical molecular weights when cut by EcoRI. Additionally, the positive control (patient with the ∆F508 mutation) will also be cut by HindIII once to produce two bands that have molecular weights of 7,200 bp and 1,800 bp. Whereas, the negative control (patient without the ∆F508 mutation) will be cut twice to produce three bands that have molecular weights of 5,700 bp, 1,800 bp, and 1,500 bp. This means that the bands that appear when Jeff’s DNA sample is cut with HindIII should be identical in weight to the patient with the ∆F508 mutation whose DNA was also cut with HindIII, and both of these samples should be different than when HindIII is used with the patient who does not have the ∆F508 mutation.

Materials

To begin, six DNA samples were obtained: two samples from the patient being tested for cystic fibrosis (Jeff), two samples from an individual with the ∆F508 mutation, and two samples from an individual without the ∆F508 mutation. The samples from the individual with the ∆F508 mutation had previously been cut with EcoRI and HindIII respectively, nothing else was added to these samples until the loading dye was added. Reaction buffer was combined with the other samples, and then EcoRI was added to one DNA sample of the individual without the ∆F508 mutation and to one of Jeff’s DNA sample. Next, HindIII was added to one DNA sample of the individual without the ∆F508 mutation and to one of Jeff’s DNA sample. These four samples were incubated at 37 degrees Celsius for thirty minutes. A solution of 0.8% agarose gel was prepared with agarose, 1x TAE buffer, and 10,000x Sybr Safe DNA gel stain. This solution was poured into a gel tray locked into a casting rack, a comb was added and then the gel was placed in a refrigerator for thirty minutes to solidify. The comb was removed and then the gel tray with solid gel was lowed into the electrophoresis chamber and submerged in 1x TAE buffer. A loading dye containing Ficol was added to each of the six DNA samples. A marker was loaded into the first well/lane to display bands of the following molecular sizes: 12,000 bp, 7,000 bp, 3,000 bp, 2,500 bp, 2,000 bp, 1,800 bp, and 1,500 bp. Then the six DNA samples that had been cut with either EcoRI or HindIII were loaded into separate wells. The cover was placed on the electrophoresis chamber so that the charge would run from the negative to the positive end, the apparatus was allowed to run for 45 minutes at 120V until the due was half-way through the gel. The gel was removed from the apparatus and viewed on the UV transilluminator next to a ruler so that the distance that each dye traveled could be measured. A standard curve graph was created with the data from the measurements of the marker. The equation that this graph generated was used to approximate the molecular size of the bands from the six DNA samples. (DeCicco-Skinner, 2019).

Results

 

Figure 1: Displaying the gel on top of the UV transilluminator next to a metric ruler. Lane 1 contained the marker and produced 7 visible bands, lane 2 contained -E (DNA of the patient without the ∆F508 mutation, cut with EcoRI), lane 3 contained +E (DNA of the patient with the ∆F508 mutation, cut with EcoRI), lane 4 contained JE (Jeff’s DNA cut with EcoRI), lane 5 contained -H (DNA of the patient without the ∆F508 mutation, cut with HindIII), lane 6 contained +H (DNA of the patient with the ∆F508 mutation, cut with HindIII), lane 7 contained JH (Jeff’s DNA cut with HindIII). Lanes 2, 3, and 4 each produced two bands that were nearly identical across the three wells. Lane 5 produced four bands. Lanes 6 and 7 produced two bands that were nearly identical.

The distance the bands traveled on the gel was measured from the distance between the well and the bottom of the band. The marker from lane 1 contained bands of several molecular weights: the 12,000 bp band traveled 21.5 mm, the 7,000 bp band traveled 26.5 mm, the 3,000 bp band traveled 29.2 mm, the 2,500 bp band traveled 34.4 mm, the 2,000 bp band traveled 37.9 mm, the 1,800 bp band traveled 40.5 mm, and the 1,500 bp band traveled 44.1 mm (Figure 1). Lane 2 was loaded with the DNA of the patient without the ∆F508 mutation that was cut with EcoRI, lane 2 displayed two bands: one traveled 28.1 mm and the other traveled 35.2 mm (Figure 1). Lane 3 was loaded with the DNA of the patient with the ∆F508 mutation that was cut with EcoRI, lane 3 had two bands as well: one traveled 27.8 mm and the other 36.1 mm (Figure 1). Lane 4 was loaded with Jeff’s DNA and had been cut with EcoRI, lane 4 also had two bands in nearly the same location as the previous two wells: one band traveled 28.0 mm and the other traveled 35.9 mm (Figure 1). Lane 5 was loaded with the DNA of the patient without the ∆F508 mutation that was cut with HindIII, lane 5 had four bands traveling 27.8 mm, 35.8 mm, 42.3 mm, and 44.1 mm (Figure 1). Lane 6 was loaded with the DNA of the patient with the ∆F508 mutation that was cut with HindIII, lane 6 had two bands traveling 26.1 mm and 43.5 mm (Figure 1). Lane 7 was loaded with Jeff’s DNA and had been cut with HindIII, lane 7 had two bands that were very similar to lane 6, traveling 26.0 mm and 43.5 mm (Figure 1).

Figure 2: Displaying the Standard Curve for CFTR ∆F508 mutation restriction digestion. The log of the molecular weight of each marker band was plotted on the y-axis, and the distance in millimeters that each band migrated from the well that the marker was loaded into was plotted on the x-axis. From this data a linear trendline was added, the equation of the line of best fit was calculated and displayed (y=-0.0391x + 4.8133), and the coefficient of determination was calculated and displayed (R^2=0.8987).

The measurements that were collected from Figure 1 were used to create the standard curve graph in Figure 2. Adding a linear trendline produced an equation of the line of best-fit (y=-0.0391x + 4.8133) and yielded a 0.8987 coefficient of determination. The distance that each band traveled for the six samples was plugged into this equation and then the antilog was taken to calculate the approximate band size.

Table 1: CFTR ∆F508 mutation restriction digestion by EcoRI and HindIII results for Jeff, patient with the CFTR ∆F508 mutation, and patient without the CFTR ∆F508 mutation.
Table 1: Displaying the expected vs observed molecular weight for the marker and six DNA samples cut with EcoRI or HindIII. The molecular weight for each sample was calculated from the standard curve equation (y=-0.0391x + 4.8133). Lanes 2, 3, and 4 had bands of very similar sizes and the values were close to what was expected. Lane 5 had more bands than were expected. Lane 6 and 7 had bands that were very similar and close to what was expected.

From the equation in Figure 2 the observed band sizes were calculated for the six DNA samples and recorded in Table 1. The observed band sizes were also calculated for the marker so that the amount of error could be quantified. Table 1 shows that the calculated molecular weight is different than the expected molecular weight, this difference is most notable when the band has more base pairs. This means that the observed band size for the DNA samples should not exactly match the expected band size and that the value will be most accurate for bands with fewer base pairs. For the three DNA samples cut with EcoRI (the patients with (+E) and without (-E) the CFTR ΔF508 mutation, and Jeff (JE)) it was expected that the EcoRI would cut the DNA strand twice to produce three bands: two bands that were 2,150 bp and one band that was 4,700 bp. However, observation of these samples revealed only two bands. Patient -E had calculated bands of 2,500 bp and 5,183 bp; patient +E had calculated bands of 2,522 bp and 5,325 bp; patient JE had calculated bands of 2,568 bp and 5,229 bp. These values are very close and are essentially identical. The patient without the CFTR ΔF508 mutation (-H) whose DNA was cut with HindIII was expected to be cut twice to produce three bands: 1,500 bp, 5,700 bp, and 1,800 bp. However, four bands were observed indicating the DNA was cut in four places. This produced bands that were calculated to be 1,227 bp, 1,443 bp, 2,591 bp, and 5,325 bp. The patient with the CFTR ΔF508 mutation (+H) and Jeff (JH) whose DNA was cut with HindIII was expected to be cut once to produce two bands that were 7,200 bp and 1,800 bp. In actuality, these two samples were cut once, in the +H patient the band sizes were 6,206 bp and 1,296 bp, whereas in the JH patient the band sizes were 6,262 bp and 1,296 bp. These values are so similar that the band sizes could be considered identical.

Discussion

With over 2000 polymorphisms of cystic fibrosis mutations, there is certainly a lot that can go wrong for the CFTR gene. In the example of the CFTR ∆F508 mutation, a single amino acid (phenylalanine) is deleted at position 508 of the gene (Suaud et al. 2011). The deletion of phenylalanine at this position is so severe that the CFTR protein becomes dysfunctional, resulting in hyperproduction of mucus across epithelial surfaces (Kreda et al. 2012). The CFTR protein has been identified through several experiments as a cyclic adenosine monophosphate-dependent phosphorylation (cAMP) activated anion channel that transports salts (chloride ions and bicarbonate ions) and other anions across epithelial cells (Gentzsch, 2018). Healthy, fully functional CFTR proteins transport these salts across the plasma membrane of epithelial cells that line the lungs and other organs to clear away excess mucus by hydrating the surface (Gentzch, 2018; Suaud et al. 2011). In the case of the CFTR ∆F508 mutation, an abnormal CFTR protein which cannot fold properly is produced; the protein cannot fold because the section of CFTR that interacts with ATP to bind nucleotides called the nucleotide-binding domain (NBDI) and the fourth cytosolic loop within another section of CFTR that anchor other protein into the plasma membrane (MSD 2) form hydrogen bonds with the arginine amino acid at the 1070 codon (Cutting, 2015). The cell detects the misfolded CFTR proteins and degrades most of them within the endoplasmic reticulum (Suaud et al. 2011). This means that very little or none of the CFTR protein reaches the surface of the epithelial cells; therefore, chloride and bicarbonate ions cannot be transported across the plasma membrane (Suaud et al. 2011). Since these salt ions cannot be transported across the plasma membrane then the salts are in higher concentration on the basal side of the epithelial cells, this draws water away from the apical surface and leads to dehydration of the lung, gastrointestinal, and pancreatic surfaces (Gentzsch, 2018). When there is no water on the apical surfaces of these organs excess mucus cannot be cleared away and then dense sticky mucus creates obstructions and leads to respiratory illnesses and inflammation that eventually cause death in patients (Gentzsch, 2018).

This experiment tested 3-year old Jeff for Cystic Fibrosis caused by the CFTR ∆F508 mutation using RFLP analysis with EcoRI and HindIII restriction endonucleases. His results were compared to a positive control (patient with the ∆F508 mutation) and a negative control (patient without the ∆F508 mutation) who also were included in the RFLP analysis with EcoRI and HindIII so that Jeff’s results can be compared to something. The results of the RFLP analysis were visualized with electrophoresis on agarose gel (Figure 1) and then an equation was produced from a standard curve (Figure 2) which was used to calculate the molecular sizes (Table 1) of the DNA fragments that the restriction endonucleases produced. This early diagnosis is critical to ensuring that Jeff receives the care he needs so he can live a painless and long life. Furthermore, RFLP analysis is a much more reliable method to test for autosomal recessive disorders than direct-to-consumers genetic tests such as 23andMeTM because there is less room for misunderstandings. Companies like 23andMeTM measure genetic variation by comparing the customer's genetic information to their database of mutations, this database can detect 715,000 single nucleotide mutations (Lu et al. 2017). This means that the database would detect the three-nucleotide deletion that occurs when phenylalanine is deleted at position 508 of the CFTR gene. RFLP is different than this because it does not compare genetic sequences, rather it uses restriction endonucleases to cut DNA into fragments at recognition sequences, in this case, cystic fibrosis could be diagnosed if the DNA of a patient was cut at the same locations as a positive control. One advantage to genetic tests like those provided by 23andMeTM is that they can also tell if a patient is a carrier of a recessive mutation, this is something that RFLP analysis cannot do (Lu et al. 2017). However, one of the largest concerns with direct-to-consumer genetic testing is that most people do not know how to interpret the results and may take drastic behaviors as a result of not receiving genetic counseling (Pare, 2012), this is another reason why it is important to test for cystic fibrosis with RFLP instead of using 23andMeTM.

It was hypothesized that Jeff has cystic fibrosis caused by the CFTR ∆F508 mutation because he displays several of the symptoms associated with the disease. This would be possible if both of his parents were carriers of the CFTR ∆F508 mutation because carriers do not have symptoms since the disease only occurs when both alleles on this location of the CFTR gene are mutated. It was predicted that when Jeff’s DNA would be cut by EcoRI two times producing three bands with the sizes 2,150 bp, 2,150 bp, and 4,700 bp and that his DNA would be cut by HindIII once producing two bands with the sizes 7,200 bp and 1,800 bp. Also, the EcoRI samples (patients with and without the ∆F508 mutation) would be cut by EcoRI two times in an identical manner to Jeff’s DNA producing three bands with the sizes 2,150 bp, 2,150 bp, and 4,700 bp. Meaning that Jeff’s DNA and the DNA of the patients with and without the ∆F508 mutation should have bands identical in size when cut by EcoRI. Additionally, the positive control (patient with the ∆F508 mutation) would also be cut by HindIII once, similar to Jeff’s DNA producing two bands with sizes 7,200 bp and 1,800 bp. This is in contrast to the negative control (patient without the ∆F508 mutation) whose DNA was predicted to be cut twice, producing three bands with sizes 5,700 bp, 1,800 bp, and 1,500 bp. If the hypothesis is correct then the bands that appear when Jeff’s DNA sample is cut with HindIII would be identical in weight to the patient with the ∆F508 mutation whose DNA was also cut with HindIII, and both of these samples should be different than when HindIII was used with the patient who does not have the ∆F508 mutation.

The purpose of including the positive control was so that the HindIII restriction digestion results of an individual with ∆F508 mutation can be visualized and then compared to how Jeff’s DNA was cut, if they were cut the same way then it means he also has the same CFTR polymorphism. If his DNA is cut differently than the positive control, then it would mean he does not have the ∆F508 mutation. The purpose of including the negative control was so that the HindIII restriction digestion results of an individual without ∆F508 mutation can be visualized and then compared to how Jeff’s DNA was cut. If the negative control was cut differently than both the positive control and Jeff’s DNA then it would mean that he does not have the ∆F508 mutation. However, if Jeff’s results did not match either the positive or negative it would mean that the results of the experiment are invalid. Also, if the positive and negative controls are identical when cut by HindIII then the experimental results would be invalid. Furthermore, even though the results of the RFLP analysis will be the same for Jeff and the controls when cutting with EcoRI, the EcoRI was still included because if this did not produce identical results for all of the samples then the results of the experiment would have to be called into question.

The marker that was loaded into lane 1 successfully moved across the gel (Figure 1) and produced bands at 12,000 bp, 7,000 bp, 3,000 bp, 2,500 bp, 2,000 bp, 1,800 bp, and 1,500 bp as was expected (Table 1). The distance that each band migrated was measured and this data was plotted against the log of each molecular weight to produce a standard curve that yielded a 0.8987 coefficient of determination and line of best fit with the equation y=-0.0391x +4.8133 (Figure 2). The distance that each band traveled for the six samples was plugged into this equation and then the antilog was taken to calculate the approximate band size. This equation was used to calculate the size of the markers as well to determine how much mathematical error is present in this model. Table 1 shows that the calculated molecular weight is slightly different than the actual molecular weight, this effect is amplified when the DNA has more base pairs. Meaning that the results of the other DNA samples could also have some variation in the expected vs observed molecular weight of the bands and that even with such variation the results would still be valid. This equation was also used to calculate the molecular sizes of the bands shown on the gel in Figure 1. If the hypothesis is correct then Jeff’s DNA, the patient with the ∆F508 mutation, and the patient without the ∆F508 mutation would all be cut twice by EcoRI to produce bands with molecular weights of 2,150 bp, 2,150 bp, and 4,700 bp. Since two of these bands are the same molecular weight then only two bands would appear on the gel: 2,150 bp and 4,700 bp. Observation of these DNA samples showed that they all have two bands (Figure 1 – lanes 2,3,4) and calculations from the standard curve equation revealed that the molecular weight of these two bands in the three samples were very similar: in JE the sizes of the DNA fragments were calculated as 2,568 bp and 5,229 bp; in -E the sizes of the bands were calculated as 2,500 bp and 5,183 bp; in +E the sizes of the bands were calculated as 2,522 bp and 5,325 bp (Table 1). Since the EcoRI cut all of the samples in approximately the same place then it can be concluded that the RFLP analysis worked correctly and that the other results of the experiment should have also worked correctly. This also means that the CFTR gene was actually present in all of the DNA samples because if any of the samples lacked the CFTR gene the samples would not have produced identical DNA fragments. However, these results alone cannot provide a diagnosis for cystic fibrosis because the EcoRI enzyme cuts the positive and negative control the same way. If EcoRI and the positive control had been the only things used to diagnose Jeff, then the hypothesis would have been accepted because the CFTR gene is cut into the same DNA fragments for the positive control and Jeff. Paradoxically, if EcoRI and the negative control had been the only things used to diagnose Jeff then the hypothesis would have been rejected because the CFTR gene is cut into the same DNA fragments for the negative control and Jeff. Obviously, this is problematic because the hypothesis cannot both be accepted and rejected, therefore, to accurately diagnosis Jeff with cystic fibrosis a restriction endonuclease that cuts the positive and negative control differently must be used, this is why HindIII was used as well.

If the hypothesis was correct then Jeff’s DNA and the (positive control) patient with the ∆F508 mutation would be cut once by HindIII to produce bands with molecular weights of 7,200 bp and 1,800 bp. Observation of these DNA samples showed that they both were cut once and had two bands (Figure 1 – lanes 6 and 7) and calculations from the standard curve equation revealed that the molecular weight of these two bands in both samples was similar: in JH the sizes of the bands were calculated as 6,262 bp and 1,296 bp; and in +H the sizes of the bands were calculated as 6,206 bp and 1,296 bp (Table 1). Since the HindIII cut both Jeff’s DNA and the DNA of the patient who has the ∆F508 mutation it is likely that Jeff also has the ∆F508 mutation which causes cystic fibrosis. To confirm that these results are valid both these samples must be compared to the negative control – the patient without the CFTR ∆F508 mutation. Observation revealed that the DNA of the patient (-H) without the ∆F508 mutation was cut three times by HindIII to produce four bands (Figure 1) and these bands were calculated to have the molecular weights of 1,227 bp, 1,443 bp, 2,591 bp, and 5,325 bp (Table 1). Obviously, these results do not match the prediction which stated that this patient’s DNA would be cut twice by HindIII to produce three bands with the molecular weights of 5,700 bp, 1,800 bp, and 1,500 bp. However, the top two bands appear to align with the bands from the samples cut with EcoRI. Meaning that this sample was likely contaminated with some EcoRI. This interferes with the analysis of the results; however, some conclusions can still be drawn due to the placement of these bands in relation to the other HindIII samples. First, the results are different enough that it can be concluded that JH and +H were identical whereas -H was different. Second, the -H sample has a band that is below the lowest band in +H and JH (Figure 1– lanes 5, 6, 7). This would make sense if the hypothesis was correct because -H should have its smallest band be 1,500 bp and +H/JH should have their smallest bands at 1,800 BP (Table 1). Third, the only way that Jeff’s DNA sample could be cut by HindIII at 7,200 bp would be if he had the CFTR ∆F508 mutation (Figure 1 and Table 1). Therefore, the hypothesis is accepted, Jeff has cystic fibrosis caused by the CFTR ∆F508 mutation.

If the positive control had been omitted then the diagnosis would be very difficult to make and the hypothesis could not be confirmed, because the calculated molecular size of Jeff’s DNA fragments is different than the expected molecular sizes. Observing that the calculated molecular size of the also differed from the expected size for the positive control’s DNA fragments and that the molecular size of these fragments and Jeff’s were nearly identical when cut by HindIII was the ultimate factor that led to accepting the hypothesis. If the negative control had been omitted, then the hypothesis would have still been accepted because Jeff’s DNA would have still matched the positive control. However, in the alternative scenario where he did not have cystic fibrosis, missing a negative control would mean that it would be impossible to confirm that Jeff did not have cystic fibrosis because his DNA fragments would not have matched anything when cut by HindIII.

This experiment only used the segment of Jeff’s genome that included the CFTR gene. If we had used his entire genome of a patient with or without the mutation then EcoRI and HindIII would have located many more recognition sites (GAATTC and AAGCTT respectively) and cleaved phosphate groups at these sites, this would produce many more DNA fragments. In fact, so many DNA fragments that it would be difficult to tell what you were looking at, Instead of seeing a few bands on Figure 1 instead there might be hundreds of bands. This would make it impossible to identify whether or not Jeff had a mutation in CFTR. The lab protocol would need to be modified if entire genomes were used so that a specific region of DNA could be examined. One way to achieve this is through southern blotting: where the DNA fragments from electrophoresis are transferred to a membrane by upward capillary transfer and then immobilized, allowing for the bands matching the CFTR sequence to be identified with a probe (Brown, 2001).

In conclusion, these findings are significant because it reveals that HindIII is a very useful restriction endonuclease for diagnosing cystic fibrosis and Jeff’s cystic fibrosis diagnosis (caused by the CFTR ∆F508 mutation) means that he can receive personalized-treatment, lumacaftor, for example, is a drug that might benefit Jeff by preventing the degradation of the misfolded CFTR proteins caused by the CFTR ∆F508 mutation, this helps the proteins reach the apical epithelial cell surface so that function can be partially restored (Kreda et al. 2012). This experiment could be improved doubling the number of samples to ensure that the samples have not been contaminated by the incorrect restriction endonuclease and by using a different trendline that produces an equation that calculates molecular sizes with improved accuracy. Future studies can explore the application of other restriction endonucleases such as BamHI, EcoRII, Hand HaeIII to see how they cut the CFTR in the positive and negative controls.

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